Gram-negative bacteria refer broadly to the bacteria which are stained in red in Gram's staining reaction, and they are different from the Gram-positive bacteria during the Gram's staining due to the difference in the structure of the cell wall (the positive bacteria being stained in violet). The Gram-negative bacteria are represented by Escherichia coli, as well as Proteus, Shigella dysenteriae, Klebsiella pneumoniae, Brucella bacilli, Haemophilus influenzae, Haemophilus parainfluenzae, Moraxella catarrhalis, Acinetobacter and so on. Pathogenicity of such bacteria is often associated with a particular component in their cell-walls, lipopolysaccharide (also known as endotoxin). In human's body, lipopolysaccharide will induce the body to produce a large amount of cytokines and to activate the immune system, eventually evoking innate immune responses against the pathogenic bacteria within the body. For instance, redness and swelling are the results of generation and release of a large amount of cytokines.
In addition to eliminating the focus of infection and a symptomatic and supportive treatment, antibiotics also need to be used during the treatment of a Gram-negative bacteria infection. At present, the antibiotics mainly used are aminoglycosides, beta-lactams, etc. These antibiotics have potent bactericidal effects on various Gram-negative bacteria, and moreover exhibit longer post-antibiotic effects on the common Gram-negative bacilli such as Pseudomonas aeruginosa, Klebsiella pneumonia, and E. coli. In addition to the antibiotics, the agents such as prostaglandin synthetase inhibitor, levamisole, and tuftsin are also useful in treatment of the Gram-negative bacteria infection. However, with the extensive application of antibiotics, the abuse of antibiotics in clinical medicine results in progression of the drug resistance of bacteria from single drug resistance into multi drug resistance, thus making many second-line antibiotics which would have been used effectively as alternatives ineffective. Secondly, the antibacterial drug may induce the generation of endotoxin while killing/inhibiting the bacteria, which increases the difficulty of treating the disease. Thus, while novel antibiotics with potent killing effect come out continuously and advanced supportive therapies occurs, treatment of the endotoxemia caused by Gram's negative bacteria infection is still a challenge in the clinical scenario, particularly with an unacceptable mortality rate of 20-30%. It has been reported that lipopolysaccharide is the major pathogenic factor which leads to a series of toxicity reaction occurring post the Gram-negative bacteria infection. Although antibiotics have a better effect of eliminating the bacteria, they have no effect on the lipopolysaccharide free in the blood and a wide variety of detrimental cytokines produced by target cells which are persistently stimulated with the free lipopolysaccharide. Therefore, when choosing an antibacterial drug clinically, a comprehensive consideration should be taken for the result of drug sensitive test and the feature of inducing release of the endotoxin. Thirdly, since lipopolysaccharide is located on the surface of the cell wall of Gram-negative bacteria, many early types of antibiotics are unable to inhibit such bacteria effectively. Based on these reasons, novel fields are being explored actively in treating Gram-negative bacteria infection in the recent years.
Antimicrobial peptides (AMPs) refer to short peptides having antibacterial activities, most of which have thermal stability, high alkalincity and broad-spectrum anti-bacterial activities. Currently, about more than or equal to 2000 of AMPs have already been identified from various organisms. These AMPs are synthesized after induction, play an important key role in an organism's resistance against the invasion of a pathogen, and are considered an important defensive constituent for the non-specific immunological function in an organism. Accordingly, it becomes a hot spot and a challenge to find out novel APMs against Gram-negative bacteria.
The EGF domain (the EGF-like domain, epidermal growth factor-like domain) is protein domains which is relatively conserved in evolution and has been designated due to first identification of the domain in epidermal growth factor. The EGF domain usually comprises 30-50 amino acid residues and has been currently identified in many animal proteins. E.g. human coagulation factor VII (human factor VII, hF VII). hF VII is a naturally-occurring protein in the human's body with molecular weight of about 50 kD. The molecule thereof comprises four domains: a membrane-bound N-terminal γ-carboxyglutamic acid domain (Gla domain), two EGF domains (EGF1 and EGF2), and a C-terminal serine protease domain. The human coagulation factor VII-EGF1 domain (hF VII-EGF1) has a molecular weight of about 3.9 kD. In human origin, there are also many analogues with the sequences similar to that of hF VII-EGF1, such as human-originated coagulation factor VII-EGF2 protein (hF VII-EGF2), human-originated coagulation factor IX-EGF1 protein (hF IX-EGF1), human-originated coagulation factor IX-EGF2 protein (hF IX-EGF2), human-originated coagulation factor X-EGF1 protein (hF X-EGF1), human-originated coagulation factor X-EGF2 protein (hF X-EGF2), etc. Up to now, there is no relevant report on treatment of a bacteria infection with a human-originated EGF domain protein as the antibacterial agent and no report on preparation of a medicament for treating endotoxemia caused by a Gram-negative bacterium.